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The efficient utilization of lignin, the direct source of renewable aromatics, into value‐added renewable chemicals is an important step towards sustainable biorefinery practices. Nevertheless, owing to the random heterogeneous structure and limited solubility, lignin utilization has been primarily limited to burning for energy. The catalytic depolymerization of lignin has been proposed and demonstrated as a viable route to sustainable biorefinery, however, low yields and poor selectivity of products, high char formation, and limited to no recycling of transition‐metal‐based catalyst involved in lignin depolymerization demands attention to enable practical‐scale lignocellulosic biorefineries. In this study, we demonstrate the catalytic depolymerization of ionic liquid‐based biorefinery poplar lignin into guaiacols over a reusable zirconium phosphate supported palladium catalyst. The essence of the study lies in the high conversion (>80 %), minimum char formation (7–16 %), high yields of guaiacols (up to 200 mg / g of lignin), and catalyst reusability. Both solid residue, liquid stream, and gaseous products were thoroughly characterized using ICP‐OES, PXRD, CHN analysis, GC‐MS, GPC, and 2D NMR to understand the hydrogenolysis pathway. [ABSTRACT FROM AUTHOR]

Lignin, the second most abundant biopolymer found in nature, has emerged as a potential source of sustainable fuels, chemicals, and materials. Finding suitable solvents, as well as technologies for efficient and affordable lignin dissolution and depolymerization, are major obstacles in the conversion of lignin to value-added products. Certain ionic liquids (ILs) are capable of dissolving and depolymerizing lignin but designing and developing an effective IL for lignin dissolution remains quite challenging. To address this issue, the COnductor-like Screening MOdel for Real Solvents (COSMO-RS) model was used to screen 5670 ILs by computing logarithmic activity coefficients (ln(γ)) and excess enthalpies (HE) of lignin, respectively. Based on the COSMO-RS computed thermodynamic properties (ln(γ) and HE) of lignin, anions such as acetate, methyl carbonate, octanoate, glycinate, alaninate, and lysinate in combination with cations like tetraalkylammonium, tetraalkylphosphonium, and pyridinium are predicted to be suitable solvents for lignin dissolution. The dissolution properties such as interaction energy between anion and cation, viscosity, Hansen solubility parameters, dissociation constants, and Kamlet–Taft parameters of selected ILs were evaluated to assess their propensity for lignin dissolution. Furthermore, molecular dynamics (MD) simulations were performed to understand the structural and dynamic properties of tetrabutylammonium [TBA]+-based ILs and lignin mixtures and to shed light on the mechanisms involved in lignin dissolution. MD simulation results suggested [TBA]+-based ILs have the potential to dissolve lignin because of their higher contact probability and interaction energies with lignin when compared to cholinium lysinate. [ABSTRACT FROM AUTHOR]

Background: Lignocellulosic resources are promising feedstocks for the manufacture of bio-based products and bioenergy. However, the inherent recalcitrance of biomass to conversion into simple sugars currently hinders the deployment of advanced bioproducts at large scale. Lignin is a primary contributor to biomass recalcitrance as it protects cell wall polysaccharides from degradation and can inhibit hydrolytic enzymes via non-productive adsorption. Several engineering strategies have been designed to reduce lignin or modify its monomeric composition. For example, expression of bacterial 3-dehydroshikimate dehydratase (QsuB) in poplar trees resulted in a reduction in lignin due to redirection of metabolic flux toward 3,4-dihydroxybenzoate at the expense of lignin. This reduction was accompanied with remarkable changes in the pools of aromatic compounds that accumulate in the biomass. Results: The impact of these modifications on downstream biomass deconstruction and conversion into advanced bioproducts was evaluated in the current study. Using ionic liquid pretreatment followed by enzymatic saccharification, biomass from engineered trees released more glucose and xylose compared to wild-type control trees under optimum conditions. Fermentation of the resulting hydrolysates using Rhodosporidium toruloides strains engineered to produce α-bisabolene, epi-isozizaene, and fatty alcohols showed no negative impact on cell growth and yielded higher titers of bioproducts (as much as + 58%) in the case of QsuB transgenics trees. Conclusion: Our data show that low-recalcitrant poplar biomass obtained with the QsuB technology has the potential to improve the production of advanced bioproducts. [ABSTRACT FROM AUTHOR]

Lignin nanoparticles (LNPs) are usually produced from lignin solution through supersaturation. The structure of the lignin in solution is still poorly understood due to structural variability of isolated lignins. Here, lignins were extracted from different plants to establish a general pattern of their structure in several lignin solvents. Lignin molecules (lignin subunits) and larger aggregates were observed in dimethyl sulfoxide (DMSO), ethylene glycol (EG) and 0.1 N NaOD solutions by small‐angle neutron scattering (SANS). It was proposed that the aggregates were composed of lignin subunits with a higher molecular weight and a higher ratio of the aliphatic to phenolic hydroxyl groups. The size, shape, and compactness are important factors that affect the uses of the LNPs, which were obtained from the SANS data for the first time. A discrepancy in the radius between SANS and DLS was discovered, pointing to a large hydration shell around the LNPs in aqueous solutions. The cytotoxicity of the corncob lignin, kraft lignin, and their LNPs were measured and compared. [ABSTRACT FROM AUTHOR]

Growing interest in sustainable sources of chemicals and energy from renewable and reliable sources has stimulated the design and synthesis of renewable Schiff-base (iminium) ionic liquids (ILs) to replace fossil-derived ILs. In this study, we report on the synthesis of three unique iminium-acetate ILs from lignin-derived aldehyde for a sustainable "future" lignocellulosic biorefinery. The synthesized ILs contained only imines or imines along with amines in their structure; the ILs with only imines group exhibited better pretreatment efficacy, achieving >89% sugar release. Various analytical and computational tools were employed to understand the pretreatment efficacy of these ILs. This is the first study to demonstrate the ease of synthesis of these renewable ILs, and therefore, opens the door for a new class of "Schiff-base ILs" for further investigation that could also be designed to be task specific. [ABSTRACT FROM AUTHOR]

The solubility parameter (SP) of a molecular species is a vital feature that indicates polarity and quantifies the 'like-seeks-like' principle, which is used in chemistry to screen solvents for dissolution. Recent studies have demonstrated that ionic liquids (ILs) and deep eutectic solvents (DESs) efficiently solubilize lignocellulosic biomass and promote enzymatic saccharification into sugars used for the production of biofuels and value-added chemicals. Understanding the solubility of plant biopolymers, particularly lignin, in ILs and DESs is critical for selecting candidate ILs and DESs for biomass pretreatment; however, experimentally measuring SPs is challenging. Thus, the present study investigates lignin dissolution mechanisms in IL/DES and prediction of the solubility parameters (Hildebrand and Hansen) of lignin, ILs, and DESs using multi-resolution simulation approaches. Solubility parameters of the studied compounds were predicted using molecular dynamics (MD) simulations, and the SP of lignin was determined to be 23–27 MPa1/2, which was close to the polymeric lignin solubility parameter (24.3–25.5 MPa1/2). The SPs of ILs namely [Ch][Lys], [Ch][Oct], and [Emim][Lys] were predicted to be ∼26 MPa1/2, which is close to lignin's SPs and resulted in increased biomass delignification. The MD simulated SPs were validated by both the COSMO-RS model and experimental investigations, with the results showing a close agreement between the predicted and experimentally obtained SPs. In addition, the enthalpy of vaporization (ΔHvap) of ILs/DESs was predicted based on the potential energy of the system, and the ΔHvap of ILs/DESs was around 40–65 kcal mol−1, which is 5–8 times higher than that of traditional organic solvents. [ABSTRACT FROM AUTHOR]

Lignin undergoes catalytic depolymerization in the presence of a variety of transfer hydrogenation agents, however the mechanisms for non-catalytic depolymerization of lignin via transfer hydrogenation are not well understood; this makes process optimization difficult. Herein, for the first time a mechanism for this process is proposed. For the purposes of understanding the mechanisms involved in these non-catalytic lignin depolymerization processes, this study investigates the equilibrium system of formic acid, methyl formate and carbon monoxide, as agents for the depolymerization of lignin, in the presence of either water or methanol as solvents. In the methyl formate/water (at 300 °C) system, 73 wt% oil was produced which contained a significant amount of low molecular weight alkylphenols, with less than 1 wt% char produced. In aqueous media, the results showed that methyl formate maintains an equilibrium with formic acid which is itself in equilibrium with carbon monoxide. It was found that using either formic acid or methyl formate for non-catalytic transfer hydrogenation of lignin can produce high amounts of oil, and can be described as a two-stage mechanism. After 10 min of reaction at 300 °C, around a quarter of the formic acid is consumed via hydride transfer of the formate proton, preventing the condensation of lignin fragments. At the same time, approximately three quarters of the formic acid decomposes to carbon dioxide and carbon monoxide. Once the formic acid is consumed, the carbon monoxide was identified as the precursor to a reactive reductive reagent and was able to activate the proton of the water molecule preventing further condensation of the lignin fragments. It has been previously thought that transfer hydrogenation in lignin using formic acid occurs via the production of molecular hydrogen. Here it is demonstrated that formic acid reacts directly with the lignin, without this hydrogen formation. Therefore the key parameters for efficient transfer hydrogenation of the lignin to maximize bio-oil yield appear to involve controlling the reactions between lignin and formic acid, methyl formate or carbon monoxide under aqueous conditions, thereby reducing the reagent cost and loading while maintaining efficient lignin conversion. [ABSTRACT FROM AUTHOR]

A fundamental understanding of lignin solubilization offers structural information that would benefit a variety of value added applications. Small angle neutron scattering (SANS) and nuclear magnetic resonance (NMR) spectroscopy were used to study correlations between the functional groups/substructures and solution structures of lignin in DMSO-d6 and 0.1 N NaOD. Three types of alkaline lignins (Sigma-Aldrich kraft lignin, poplar wood kraft lignin, and corncob soda lignin), exhibiting different degrees of aggregation in 0.4 wt% solutions, were investigated to identify the major intermolecular interactions that cause lignin aggregation. Intermolecular hydrogen bonding, non-covalent π–π interactions between phenyl rings, lignin chain conformation and the degree of branching were discussed. Different operating forces for lignin solubilization and aggregation were found in DMSO-d6 and NaOD solutions. [ABSTRACT FROM AUTHOR]

Lignin in plant biomass represents a target for engineering strategies towards the development of a sustainable bioeconomy. In addition to the conventional lignin monomers, namely p-coumaryl, coniferyl and sinapyl alcohols, tricin has been shown to be part of the native lignin polymer in certain monocot species. Because tricin is considered to initiate the polymerization of lignin chains, elucidating its biosynthesis and mechanism of export to the cell wall constitute novel challenges for the engineering of bioenergy crops. Late steps of tricin biosynthesis require two methylation reactions involving the pathway intermediate selgin. It has recently been demonstrated in rice and maize that caffeate O-methyltransferase (COMT) involved in the synthesis syringyl (S) lignin units derived from sinapyl alcohol also participates in the synthesis of tricin in planta. In this work, we validate in sorghum (Sorghum bicolor L.) that the O-methyltransferase responsible for the production of S lignin units (SbCOMT / Bmr12) is also involved in the synthesis of lignin-linked tricin. In particular, we show that biomass from the sorghum bmr12 mutant contains lower level of tricin incorporated into lignin, and that SbCOMT can methylate the tricin precursors luteolin and selgin. Our genetic and biochemical data point toward a general mechanism whereby COMT is involved in the synthesis of both tricin and S lignin units. [ABSTRACT FROM AUTHOR]

Background: Biomass pretreatment remains an essential step in lignocellulosic biofuel production, largely to facilitate the efficient removal of lignin and increase enzyme accessibility to the polysaccharides. In recent years, there have been significant efforts in planta to reduce lignin content or modify its composition to overcome the inherent recalcitrance that it imposes on lignocellulosic biomass during processing. Here, transgenic poplar lines in which monolignol ferulate conjugates were synthesized during cell wall development to introduce, during lignification, readily cleavable ester linkages into the lignin polymer backbone (i.e., "zip lignin"), along with wild-type (WT) controls, were pretreated with different ionic liquids (ILs). Results: The strategic introduction of ester bonds into the lignin backbone resulted in increased pretreatment efficiency and released more carbohydrates with lower energy input. After pretreatment with any of three different ILs, and after limited saccharification, the transgenic poplars, especially those with relatively higher amounts of incorporated monolignol ferulate conjugates, yielded up to 23% higher sugar levels compared to WT plants. Conclusion: Our findings clearly demonstrate that the introduction of ester linkages into the lignin polymer backbone decreases biomass recalcitrance in poplar has the potential to reduce the energy and/or amount of IL required for effective pretreatment, and could enable the development of an economically viable and sustainable biorefinery process. [ABSTRACT FROM AUTHOR]

The relatively poor solubility of lignin in most pretreatment solvents remains one of the biggest challenges in lignin valorization to improve overall biorefinery economics. In this work, rapid room temperature solubilization of lignin at high solid loadings (＞30 wt%) can be easily achieved in a single step using ethylene glycol (EG). The solubilized lignin can be rapidly and quantitatively recovered with the addition of ethanol. The computational and nuclear magnetic resonance (NMR) spectroscopic studies confirm that strong hydrogen bond interactions between EG and the free hydroxyl groups present in lignin contribute to the lignin dissolution. In addition, hydrogen peroxide mediated depolymerization of the dissolved lignin at a low temperature (80 °C) was tested and the effect of EG molecules on depolymerization of lignin was also theoretically studied. The findings of this work provide mechanistic insights of hydrogen bond interactions in high lignin solubilization and valorization. [ABSTRACT FROM AUTHOR]

Sphingobium sp. SYK-6 is a soil bacterium boasting a well-studied ligninolytic pathway and the potential for development into a microbial chassis for lignin valorization. An improved understanding of its metabolism will help researchers in the engineering of SYK-6 for the production of value-added chemicals through lignin valorization. We used 13C-fingerprinting, 13C metabolic flux analysis (13C-MFA), and RNA-sequencing differential expression analysis to uncover the following metabolic traits: (i) SYK-6 prefers alkaline conditions, making it an efficient host for the consolidated bioprocessing of lignin, and it also lacks the ability to metabolize sugars or organic acids; (ii) the CO2 release (i.e., carbon loss) from the ligninolysis-based metabolism of SYK-6 is significantly greater than the CO2 release from the sugar-basedmetabolism of Escherichia coli; (iii) the vanillin catabolic pathway (which is the converging point of majority of the lignin catabolic pathways) is coupled with the tetrahydrofolate-dependent C1 pathway that is essential for the biosynthesis of serine, histidine, and methionine; (iv) catabolic end products of lignin (pyruvate and oxaloacetate) must enter the tricarboxylic acid (TCA) cycle first and then use phosphoenolpyruvate carboxykinase to initiate gluconeogenesis; and (v) 13C-MFA together with RNA-sequencing differential expression analysis establishes the vanillin catabolic pathway as the major contributor of NAD(P)H synthesis. Therefore, the vanillin catabolic pathway is essential for SYK-6 to obtain sufficient reducing equivalents for its healthy growth; cosubstrate experiments support this finding. This unique energy feature of SYK-6 is particularly interesting because most heterotrophs rely on the transhydrogenase, the TCA cycle, and the oxidative pentose phosphate pathway to obtain NADPH. [ABSTRACT FROM AUTHOR]

Lignin plays important biological functions in plant cell walls, but also contributes to the recalcitrance of the walls to deconstruction. In recent years, genetic modification of lignin biosynthesis pathways has become one of the primary targets of plant cell wall engineering. In this study, we used a combination of approaches to characterize the structural and compositional features of wild-type Arabidopsis and mutants with distinct lignin monomer compositions: fah1-2 (Guaiacyl, G-lignin dominant), C4H-F5H (Syringyl, S-lignin dominant), COMT1 (G/5-hydroxy G-lignin dominant), and a newly developed med5a med5b ref8 (p-hydroxyphenyl, H-lignin dominant) mutant. In order to understand how lignin modification affects biomass recalcitrance, substrate reactivity and lignin fractionation, we correlated these properties with saccharification efficiency after ionic liquid (IL) pretreatment. Results showed that the cleavage of β-O-4 linkages in the H- or S-lignin mutants was greater than that in G-lignin mutants. Furthermore, density functional theory (DFT) based calculations indicate higher chemical reactivity of the linkages between H- and S-lignin monomers, a possible cause of the reduced recalcitrance of H- or S-lignin mutants. Glycome profiling was conducted to study the impact of lignin modification on overall composition, extractability, integrity and lignin-associated features of most major non-cellulosic cell wall glycans in these mutants. This study provides insights into the role of lignin monomer composition on the enzymatic digestibility of biomass and the effect of lignin modification on overall wall structure and biomass pretreatment performance. [ABSTRACT FROM AUTHOR]

Lignin confers recalcitrance to plant biomass used as feedstocks in agro-processing industries or as source of renewable sugars for the production of bioproducts. The metabolic steps for the synthesis of lignin building blocks belong to the shikimate and phenylpropanoid pathways. Genetic engineering efforts to reduce lignin content typically employ gene knockout or gene silencing techniques to constitutively repress one of these metabolic pathways. Recently, new strategies have emerged offering better spatiotemporal control of lignin deposition, including the expression of enzymes that interfere with the normal process for cell wall lignification. In this study, we report that expression of a 3-dehydroshikimate dehydratase (QsuB from Corynebacterium glutamicum) reduces lignin deposition in Arabidopsis cell walls. QsuB was targeted to the plastids to convert 3-dehydroshikimate - an intermediate of the shikimate pathway - into protocatechuate. Compared to wild-type plants, lines expressing QsuB contain higher amounts of protocatechuate, p-coumarate, p-coumaraldehyde and p-coumaryl alcohol, and lower amounts of coniferaldehyde, coniferyl alcohol, sinapaldehyde and sinapyl alcohol. 2D-NMR spectroscopy and pyrolysis-gas chromatography/mass spectrometry (pyro-GC/MS) reveal an increase of p-hydroxyphenyl units and a reduction of guaiacyl units in the lignin of QsuB lines. Size-exclusion chromatography indicates a lower degree of lignin polymerization in the transgenic lines. Therefore, our data show that the expression of QsuB primarily affects the lignin biosynthetic pathway. Finally, biomass from these lines exhibits more than a twofold improvement in saccharification efficiency. We conclude that the expression of QsuB in plants, in combination with specific promoters, is a promising gain-of-function strategy for spatiotemporal reduction of lignin in plant biomass. [ABSTRACT FROM AUTHOR]

Genes associated with elevated oxidative enzyme activities in arid systems have not been well characterized. To link measured oxidative activities with specific enzymes, we assembled protein-coding reads from the rhizospheres (RHZ) of two arid land grasses. Targeted gene scans for open reading frames, encoding genes potentially involved in lignin modification, resulted in 127 distinct assembly products. The putative genes included those significantly similar to Class II secretory fungal peroxidases. These genes are expressed at sufficiently high levels for assembly, annotation and differentiation across experimental conditions, and they demonstrate the interplay of root systems, environment and plant microbiomes. The genes assembled also included copper-dependent lytic polysaccharide monooxygenases. We detail the enzymes in the host grass RHZs and present a preliminary taxonomic microhabitat characterization. Our findings provide support for biologically mediated Fenton chemistry in the root zones of desert grasses, and provide insight into arid land carbon flow. These results also demonstrate a hyperdiverse microbial community. Both ribosomal RNA and messenger RNA sequences were dominated by bacteria, followed by fungal sequence abundance. Among the notable fungal sequences were those from the members of the arbuscular mycorrhizal fungi (Glomeromycota), which though abundant in this study, we rarely observed in previous PCR-based surveys. [ABSTRACT FROM AUTHOR]

We report a new assay for breakdown of high molecular weight, insoluble lignin based on lignin films. In this method, decrease in film thickness is detected upon solubilization of mass through either chemical alteration of the lignin or molecular weight reduction. The assay was performed with organosolv lignin, the only chemical modification being an oxidative pretreatment to provide film stability with respect to dissolution. The assay is sensitive to release of as little as 20 Å of material from the film. A multiplexed format was developed using a silicone block in the form of a standard 96-well plate, allowing simultaneous assaying of a large number of reaction conditions. The assay was demonstrated using the Fenton reaction, revealing new insights into the physicochemical aspects of this reaction system with insoluble lignin. In particular, mass solubilized from the film was found to pass through a maximum as a function of the initial concentration of FeCl2 ([FeCl2]o), with the maximum occurring at [FeCl2]o = 1 mM for [H2O2]o = 5%. At that condition, solubilization of mass occurs in two stages. The reaction produces mostly ring-opened products of mass greater than 700 g mol−1, along with a minority of low molecular weight aromatics. The new insight from this work is an important step toward optimizing this complex reaction system for effective lignin breakdown. [ABSTRACT FROM AUTHOR]

As the attraction of creating biofuels and bio-based chemicals from lignocellulosic biomass has increased, researchers have been challenged with developing a better understanding of lignin structure, quantity and potential uses. Lignin has frequently been considered a waste-product from the deconstruction of plant cell walls, in attempts to isolate polysaccharides that can be hydrolyzed and fermented into fuel or other valuable commodities. In order to develop useful applications for lignin, accurate analytical instrumentation and methodologies are required to qualitatively and quantitatively assess, for example, what the structure of lignin looks like or how much lignin comprises a specific feedstock׳s cellular composition. During the past decade, various diverse strategies have been employed to elucidate the structure and composition of lignin. These techniques include using two-dimensional nuclear magnetic resonance to resolve overlapping spectral data, measuring biomass with vibrational spectroscopy to enable modeling of lignin content or monomeric ratios, methods to probe and quantify the linkages between lignin and polysaccharides, or refinements of established methods to provide higher throughput analyses, less use of consumables, etc. This review seeks to provide a comprehensive overview of many of the advancements achieved in evaluating key lignin attributes. Emphasis is placed on research endeavored in the last decade. [ABSTRACT FROM AUTHOR]

Breeding has transformed wild plant species into modern crops, increasing the allocation of their photosynthetic assimilate into grain, fiber, and other products for human use. Despite progress in increasing the harvest index, much of the biomass of crop plants is not utilized. Potential uses for the large amounts of agricultural residues that accumulate are animal fodder or bioenergy, though these may not be economically viable without additional efforts such as targeted breeding or improved processing. We characterized leaf and stem tissue from a diverse set of rice genotypes (varieties) grown in two environments (greenhouse and field) and report bioenergy-related traits across these variables. Among the 16 traits measured, cellulose, hemicelluloses, lignin, ash, total glucose, and glucose yield changed across environments, irrespective of the genotypes. Stem and leaf tissue composition differed for most traits, consistent with their unique functional contributions and suggesting that they are under separate genetic control. Plant variety had the least influence on the measured traits. High glucose yield was associated with high total glucose and hemicelluloses, but low lignin and ash content. Bioenergy yield of greenhouse-grown biomass was higher than field-grown biomass, suggesting that greenhouse studies overestimate bioenergy potential. Nevertheless, glucose yield in the greenhouse predicts glucose yield in the field ( ρ = 0.85, p < 0.01) and could be used to optimize greenhouse (GH) and field breeding trials. Overall, efforts to improve cell wall composition for bioenergy require consideration of production environment, tissue type, and variety. [ABSTRACT FROM AUTHOR]

Alcohols of increasing alkyl chain length were investigated as precipitants in an ionic liquid (IL) pretreatment system. Switchgrass samples pretreated by 1-ethyl-3-methylimidazolium acetate were characterized after the use of different alkyl chain lengths of alcohols as antisolvents. The resulting IL-pretreated switchgrass (PSG) samples were characterized by enzymatic hydrolysis, cross polarization/magic angle spinning (CP/MAS) C nuclear magnetic resonance (NMR), Fourier transform infrared spectroscopy (FTIR), and 2D NMR spectroscopy. Glucan digestibilities of PSG samples were ∼80 % after 72 h at 5 mg protein g glucan regardless of the antisolvent used. The use of 1-octanol as an antisolvent, with 10 % water to allow for use of wet biomass, enabled a partial lignin fractionation and multiphase separation for the IL recycle without compromising the chemical structure of the carbohydrates and lignin from the PSG. Lignin fragments were observed in the IL after pretreatment by gel permeation chromatography (GPC). After separation, both the IL and the octanol antisolvent were reused for switchgrass pretreatment and precipitation for an additional 3 cycles. The PSG samples derived from recycled IL were rapidly hydrolyzed, and a high glucan digestibility of 80 % was obtained even at a low enzyme loading of 5 mg protein g glucan. 2D NMR analysis of residual solids of PSG post-enzymatic hydrolysis revealed that lignin in these residual solids was depolymerized. This strategy enables an ease in separation of pretreated lignocellulosic solids, reduced water use, and recycle of both IL and the antisolvent. [ABSTRACT FROM AUTHOR]

Background: Lignocellulosic biomass has the potential to be a major source of renewable sugar for biofuel production. Before enzymatic hydrolysis, biomass must first undergo a pretreatment step in order to be more susceptible to saccharification and generate high yields of fermentable sugars. Lignin, a complex, interlinked, phenolic polymer, associates with secondary cell wall polysaccharides, rendering them less accessible to enzymatic hydrolysis. Herein, we describe the analysis of engineered Arabidopsis lines where lignin biosynthesis was repressed in fiber tissues but retained in the vessels, and polysaccharide deposition was enhanced in fiber cells with little to no apparent negative impact on growth phenotype. Results: Engineered Arabidopsis plants were treated with the ionic liquid (IL) 1-ethyl-3-methylimidazolium acetate 1-ethyl-3-methylimidazolium acetate ([C2C1im][OAc]) at 10 % wt biomass loading at either 70 °C for 5 h or 140 °C for 3 h. After pretreatment at 140 °C and subsequent saccharification, the relative peak sugar recovery of ~26.7 g sugar per 100 g biomass was not statistically different for the wild type than the peak recovery of ~25.8 g sugar per 100 g biomass for the engineered plants (84 versus 86 % glucose from the starting biomass). Reducing the pretreatment temperature to 70 °C for 5 h resulted in a significant reduction in the peak sugar recovery obtained from the wild type to 16.2 g sugar per 100 g biomass, whereas the engineered lines with reduced lignin content exhibit a higher peak sugar recovery of 27.3 g sugar per 100 g biomass and 79 % glucose recoveries. Conclusions: The engineered Arabidopsis lines generate high sugar yields after pretreatment at 70 °C for 5 h and subsequent saccharification, while the wild type exhibits a reduced sugar yield relative to those obtained after pretreatment at 140 °C. Our results demonstrate that employing cell wall engineering efforts to decrease the recalcitrance of lignocellulosic biomass has the potential to drastically reduce the energy required for effective pretreatment. [ABSTRACT FROM AUTHOR]

The productivity of plants as biofuel or biomaterial crops is established by both the yield of plant biomass per unit area of land and the efficiency of conversion of the biomass to biofuel. Higher yielding biofuel crops with increased conversion efficiencies allow production on a smaller land footprint minimizing competition with agriculture for food production and biodiversity conservation. Plants have traditionally been domesticated for food, fibre and feed applications. However, utilization for biofuels may require the breeding of novel phenotypes, or new species entirely. Genomics approaches support genetic selection strategies to deliver significant genetic improvement of plants as sources of biomass for biofuel manufacture. Genetic modification of plants provides a further range of options for improving the composition of biomass and for plant modifications to assist the fabrication of biofuels. The relative carbohydrate and lignin content influences the deconstruction of plant cell walls to biofuels. Key options for facilitating the deconstruction leading to higher monomeric sugar release from plants include increasing cellulose content, reducing cellulose crystallinity, and/or altering the amount or composition of noncellulosic polysaccharides or lignin. Modification of chemical linkages within and between these biomass components may improve the ease of deconstruction. Expression of enzymes in the plant may provide a cost-effective option for biochemical conversion to biofuel. [ABSTRACT FROM AUTHOR]

Background: The development of affordable woody biomass feedstocks represents a significant opportunity in the development of cellulosic biofuels. Primary woodchips produced by forest mills are considered an ideal feedstock, but the prices they command on the market are currently too expensive for biorefineries. In comparison, forestry residues represent a potential low-cost input but are considered a more challenging feedstock for sugar production due to complexities in composition and potential contamination arising from soil that may be present. We compare the sugar yields, changes in composition in Douglas-fir woodchips and forestry residues after pretreatment using ionic liquids and enzymatic saccharification in order to determine if this approach can efficiently liberate fermentable sugars. Results: These samples were either mechanically milled through a 2 mm mesh or pretreated as received with the ionic liquid (IL) 1-ethyl-3-methylimidazolium acetate [C2mim][OAc] at 120°C and 160°C. IL pretreatment of Douglas-fir woodchips and forestry residues resulted in approximately 71-92% glucose yields after enzymatic saccharification. X-ray diffraction (XRD) showed that the pretreated cellulose was less crystalline after IL pretreatment as compared to untreated control samples. Two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) revealed changes in lignin and hemicellulose structure and composition as a function of pretreatment. Mass balances of sugar and lignin streams for both the Douglas-fir woodchips and forestry residues throughout the pretreatment and enzymatic saccharification processes are presented. Conclusions: While the highest sugar yields were observed with the Douglas-fir woodchips, reasonably high sugar yields were obtained from forestry residues after ionic liquid pretreatment. Structural changes to lignin, cellulose and hemicellulose in the woodchips and forestry residues of Douglas-fir after [C2mim][OAc] pretreatment are analyzed by XRD and 2D-NMR, and indicate that significant changes occurred. Irrespective of the particle sizes used in this study, ionic liquid pretreatment successfully allowed high glucose yields after enzymatic saccharification. These results indicate that forestry residues may be a more viable feedstock than previously thought for the production of biofuels. [ABSTRACT FROM AUTHOR]

Background: Lignin is often overlooked in the valorization of lignocellulosic biomass, but lignin-based materials and chemicals represent potential value-added products for biorefineries that could significantly improve the economics of a biorefinery. Fluctuating crude oil prices and changing fuel specifications are some of the driving factors to develop new technologies that could be used to convert polymeric lignin into low molecular weight lignin and or monomeric aromatic feedstocks to assist in the displacement of the current products associated with the conversion of a whole barrel of oil. We present an approach to produce these chemicals based on the selective breakdown of lignin during ionic liquid pretreatment. Results: The lignin breakdown products generated are found to be dependent on the starting biomass, and significant levels were generated on dissolution at 160°C for 6 hrs. Guaiacol was produced on dissolution of biomass and technical lignins. Vanillin was produced on dissolution of kraft lignin and eucalytpus. Syringol and allyl guaiacol were the major products observed on dissolution of switchgrass and pine, respectively, whereas syringol and allyl syringol were obtained by dissolution of eucalyptus. Furthermore, it was observed that different lignin-derived products could be generated by tuning the process conditions. Conclusions: We have developed an ionic liquid based process that depolymerizes lignin and converts the low molecular weight lignin fractions into a variety of renewable chemicals from biomass. The generated chemicals (phenols, guaiacols, syringols, eugenol, catechols), their oxidized products (vanillin, vanillic acid, syringaldehyde) and their easily derivatized hydrocarbons (benzene, toluene, xylene, styrene, biphenyls and cyclohexane) already have relatively high market value as commodity and specialty chemicals, green building materials, nylons, and resins. [ABSTRACT FROM AUTHOR]

Lignocellulosic biomass is utilized as a renewable feedstock in various agro-industrial activities. Lignin is an aromatic, hydrophobic and mildly branched polymer integrally associated with polysaccharides within the biomass, which negatively affects their extraction and hydrolysis during industrial processing. Engineering the monomer composition of lignins offers an attractive option towards new lignins with reduced recalcitrance. The presented work describes a new strategy developed in Arabidopsis for the overproduction of rare lignin monomers to reduce lignin polymerization degree (DP). Biosynthesis of these 'DP reducers' is achieved by expressing a bacterial hydroxycinnamoyl-CoA hydratase-lyase (HCHL) in lignifying tissues of Arabidopsis inflorescence stems. HCHL cleaves the propanoid side-chain of hydroxycinnamoyl-CoA lignin precursors to produce the corresponding hydroxybenzaldehydes so that plant stems expressing HCHL accumulate in their cell wall higher amounts of hydroxybenzaldehyde and hydroxybenzoate derivatives. Engineered plants with intermediate HCHL activity levels show no reduction in total lignin, sugar content or biomass yield compared with wild-type plants. However, cell wall characterization of extract-free stems by thioacidolysis and by 2D-NMR revealed an increased amount of unusual C6C1 lignin monomers most likely linked with lignin as end-groups. Moreover the analysis of lignin isolated from these plants using size-exclusion chromatography revealed a reduced molecular weight. Furthermore, these engineered lines show saccharification improvement of pretreated stem cell walls. Therefore, we conclude that enhancing the biosynthesis and incorporation of C6C1 monomers ('DP reducers') into lignin polymers represents a promising strategy to reduce lignin DP and to decrease cell wall recalcitrance to enzymatic hydrolysis. [ABSTRACT FROM AUTHOR]

The article focuses on the use of ionic liquids as lignocellulostic biomass solvents for pretreatment methods. It states that ionic liquid pretreatment generates more efficient polysaccharide-enriched substrate and be used in mild thermal conditions. It also discusses the issues concerning the application of ionic liquid pretreatment as a commercial process including the cost of ionic liquids, the thermostability of the ionic liquid, and the removal of lignin and plant cell constituents.

Background: In this work, three pretreatments under investigation at the DOE Bioenergy Research Centers (BRCs) were subjected to a side-by-side comparison to assess their performance on model bioenergy hardwoods (a eucalyptus and a hybrid poplar). These include co-solvent-enhanced lignocellulosic fractionation (CELF), pretreatment with an ionic liquid using potentially biomass-derived components (cholinium lysinate or [Ch][Lys]), and two-stage Cu-catalyzed alkaline hydrogen peroxide pretreatment (Cu-AHP). For each of the feedstocks, the pretreatments were assessed for their impact on lignin and xylan solubilization and enzymatic hydrolysis yields as a function of enzyme loading. Lignins recovered from the pretreatments were characterized for polysaccharide content, molar mass distributions, β-aryl ether content, and response to depolymerization by thioacidolysis. Results: All three pretreatments resulted in significant solubilization of lignin and xylan, with the CELF pretreatment solubilizing the majority of both biopolymer categories. Enzymatic hydrolysis yields were shown to exhibit a strong, positive correlation with the lignin solubilized for the low enzyme loadings. The pretreatment-derived solubles in the [Ch][Lys]-pretreated biomass were presumed to contribute to inhibition of enzymatic hydrolysis in the eucalyptus as a substantial fraction of the pretreatment liquor was carried forward into hydrolysis for this pretreatment. The pretreatment-solubilized lignins exhibited significant differences in polysaccharide content, molar mass distributions, aromatic monomer yield by thioacidolysis, and β-aryl ether content. Key trends include a substantially higher polysaccharide content in the lignins recovered from the [Ch][Lys] pretreatment and high β-aryl ether contents and aromatic monomer yields from the Cu-AHP pretreatment. For all lignins, the 13C NMR-determined β-aryl ether content was shown to be correlated with the monomer yield with a second-order functionality. Conclusions: Overall, it was demonstrated that the three pretreatments highlighted in this study demonstrated uniquely different functionalities in reducing biomass recalcitrance and achieving higher enzymatic hydrolysis yields for the hybrid poplar while yielding a lignin-rich stream that may be suitable for valorization. Furthermore, modification of lignin during pretreatment, particularly cleavage of β-aryl ether bonds, is shown to be detrimental to subsequent depolymerization. [ABSTRACT FROM AUTHOR]

Background: Engineering strategies to create promoters that are both higher strength and tunable in the presence of inexpensive compounds are of high importance to develop metabolic engineering technologies that can be commercialized. Lignocellulosic biomass stands out as the most abundant renewable feedstock for the production of biofuels and chemicals. However, lignin a major polymeric component of the biomass is made up of aromatic units and remains as an untapped resource. Novel synthetic biology tools for the expression of heterologous proteins are critical for the effective engineering of a microbe to valorize lignin. This study demonstrates the first successful attempt in the creation of engineered promoters that can be induced by aromatics present in lignocellulosic hydrolysates to increase heterologous protein production. Results: A hybrid promoter engineering approach was utilized for the construction of phenolic-inducible promoters of higher strength. The hybrid promoters were constructed by replacing the spacer region of an endogenous promoter, PemrR present in E. coli that was naturally inducible by phenolics. In the presence of vanillin, the engineered promoters Pvtac, Pvtrc, and Pvtic increased protein expression by 4.6-, 3.0-, and 1.5-fold, respectively, in comparison with a native promoter, PemrR. In the presence of vanillic acid, Pvtac, Pvtrc, and Pvtic improved protein expression by 9.5-, 6.8-, and 2.1-fold, respectively, in comparison with PemrR. Among the cells induced with vanillin, the emergence of a sub-population constituting the healthy and dividing cells using flow cytometry was observed. The analysis also revealed this smaller sub-population to be the primary contributor for the increased expression that was observed with the engineered promoters. Conclusions: This study demonstrates the first successful attempt in the creation of engineered promoters that can be induced by aromatics to increase heterologous protein production. Employing promoters inducible by phenolics will provide the following advantages: (1) develop substrate inducible systems; (2) lower operating costs by replacing expensive IPTG currently used for induction; (3) develop dynamic regulatory systems; and (4) provide flexibility in operating conditions. The flow cytometry findings strongly suggest the need for novel approaches to maintain a healthy cell population in the presence of phenolics to achieve increased heterologous protein expression and, thereby, valorize lignin efficiently. [ABSTRACT FROM AUTHOR]

Lignin is the primary contributor to the high cost of biofuel-production from lignocellulosic biomass. In order to study lignin removal and the release of aromatic monomers, we applied hydrocracking and ionic liquid pretreatments on Arabidopsis thaliana biomass from both wild type (WT) and a mutant (CAD cxd) defective in two cinnamyl alcohol dehydrogenase genes involved in the lignin biosynthetic pathway. For Arabidopsis WT , our results highlight that pretreatments reduce average molecular weight of lignin by about 65% and decrease the content of β-O-4 linkages between lignin monomers. For Arabidopsis CAD mutant, an opposite effect is evidenced. Fewer differences were observed on depolymerization and molecular structure of lignin, which indicates that (8-O-4), (8-5), and (8-8) linkages observed in CAD mutant make lignin more resilient to pretreatment than wild-type lignin. Finally, our study shows the potential of hydrocracking pretreatment technology for extracting valuable aldehyde monomers such as vanillin and syringaldehyde from biomass. • Hydrocracking and ionic liquids treatments reduce average molecular weight and β-O-4 linkages of Arabidopsis thaliana wild-type. • Fewer differences were observed on Arabidopsis thaliana CAD mutant lignin after pretreatments application. • (8-O-4), (8-5), and (8-8) linkages observed in CAD mutant make lignin more resilient to pretreatment than WT lignin. • Hydrocracking allow to extract valuable aldehyde monomers from Arabidopsis WT and CAD lignin. [ABSTRACT FROM AUTHOR]

Review: 76 refs. [ABSTRACT FROM AUTHOR]

Corymbia (a eucalypt) is an important forestry genus and a potential lignocellulosic bioenergy feedstock. The composition of the lignocellulosic cell wall significantly impacts pretreatment efficiency and conversion to biofuel but is variable and changes with age. In this study, we estimated Klason lignin content, composition, and monosaccharide (glucose and xylose) release after enzymatic saccharification of untreated and hydrothermally pretreated biomass from Corymbia parental species Corymbia torelliana (CT), Corymbia citriodora subsp. variegata (spotted gum; CCV), and interspecific F1 hybrids (CT × CCV) at ages 9 and 12 years from planting. Analysis of lignin composition derived from syringyl/guaiacyl monolignols (S/G) found significant differences among taxa, with CT S/G ratios (2.2 and 2.0) being significantly lower than CCV (2.6 and 2.3) or hybrids (2.5 and 2.3) at ages 9 and 12 respectively. In general, enzymatic saccharification yields from untreated biomass were significantly different among taxa, with CT (113 and 75 mg g −1 ) and hybrids (108 and 81 mg g −1 ) yielding significantly higher glucose from untreated biomass than CCV (82 and 56 mg g −1 ) at ages 9 and 12 respectively. Comparison of traits within taxa between ages 9 and 12 found S/G ratios and glucose yields from untreated biomass were significantly lower in CT, CCV and hybrid taxa. In conclusion, the formation of lignocellulosic cell walls is complex, influenced by genetics and age of material, requiring optimization of rotation age for biofuel production and other industrial processes. [ABSTRACT FROM AUTHOR]

Microbial communities that deconstruct plant biomass have broad relevance in biofuel production and global carbon cycling. Biomass pretreatments reduce plant biomass recalcitrance for increased efficiency of enzymatic hydrolysis. We exploited these chemical pretreatments to study how thermophilic bacterial consortia adapt to deconstruct switchgrass (SG) biomass of various compositions. Microbial communities were adapted to untreated, ammonium fiber expansion (AFEX)-pretreated, and ionicliquid (IL)-pretreated SG under aerobic, thermophilic conditions using green waste compost as the inoculum to study biomass deconstruction by microbial consortia. After microbial cultivation, gravimetric analysis of the residual biomass demonstrated that both AFEX and IL pretreatment enhanced the deconstruction of the SG biomass approximately 2-fold. Two-dimensional nuclear magnetic resonance (2D-NMR) experiments and acetyl bromide-reactive-lignin analysis indicated that polysaccharide hydrolysis was the dominant process occurring during microbial biomass deconstruction, and lignin remaining in the residual biomass was largely unmodified. Small-subunit (SSU) rRNA gene amplicon libraries revealed that although the dominant taxa across these chemical pretreatments were consistently represented by members of the Firmicutes, the Bacteroidetes, and Deinococcus- Thermus, the abundance of selected operational taxonomic units (OTUs) varied, suggesting adaptations to the different substrates. Combining the observations of differences in the community structure and the chemical and physical structure of the biomass, we hypothesize specific roles for individual community members in biomass deconstruction. [ABSTRACT FROM AUTHOR]

Ionic liquids (ILs) have shown great potential for the reduction of lignin in biomass after pretreatment. Although dilute acid and base pretreatments have been shown to result in pretreated biomass with substantially different lignin composition, there is scarce information on the composition of lignin of IL pretreated biomass. In this work, temperature dependent compositional changes in lignin after IL pretreatment were studied to develop a mechanistic understanding of the process. Panicum virgatum and Eucalyptus globulus were pretreated with 1-ethyl-3-methylimidazolium acetate ([C 2 mim][OAc]). Measurement of syringyl and guaiacyl ratio using pyrolysis–GC/MS and Kamlet–Taft properties of [C 2 mim][OAc] at 120 °C and 160 °C strongly suggest two different modes of IL pretreatment. Preferential breakdown of S-lignin in both eucalyptus and switchgrass at high pretreatment temperature (160 °C) and breakdown of G-lignin for eucalyptus and no preferential break down of either S- or G-lignin in switchgrass was observed at lower pretreatment temperatures (120 °C). [ABSTRACT FROM AUTHOR]

The solution structures of three types of isolated ligninorganosolv(OS), Kraft (K), and low sulfonate (LS)before and after treatmentwith 1-ethyl-3-methylimidazolium acetate were studied using small-angleneutron scattering (SANS) and dynamic light scattering (DLS) overa concentration range of 0.3–2.4 wt %. The results indicatethat each of these lignins is comprised of aggregates of well-definedbasal subunits, the shapes and sizes of which, in D2O andDMSO-d6, are revealed using these techniques.LS lignin contains a substantial amount of nanometer-scale individualsubunits. In aqueous solution these subunits have a well-defined elongatedshape described well by ellipsoidal and cylindrical models. At lowconcentration the subunits are highly expanded in alkaline solution,and the effect is screened with increasing concentration. OS lignindissolved in DMSO was found to consist of a narrow distribution ofaggregates with average radius 200 ± 30 nm. K lignin in DMSOconsists of aggregates with a very broad size distribution. Afterionic liquid (IL) treatment, LS lignin subunits in alkaline solutionmaintained the elongated shape but were reduced in size. IL treatmentof OS and K lignins led to the release of nanometer-scale subunitswith well-defined size and shape. [ABSTRACT FROM AUTHOR]

• A general overview of recent development and challenges of IL pretreatment technologies. • Reinforced pretreatment techniques using dialkylimidazolium-based ILs. • Choline-based ILs with better biocompatibility. • Low-cost protic acidic ILs. Processes that can convert lignocellulosic biomass into biofuels and chemicals are particularly attractive considering renewability and minimal environmental impact. Ionic liquids (ILs) have been used as novel solvents in the process development in that they can effectively deconstruct recalcitrant lignocellulosic biomass for high sugar yield and lignin recovery. From cellulose-dissolving ILs to choline-based and protic acidic ILs, extensive research in this field has been done, driven by the promising future of IL pretreatment. Meanwhile, shortcomings and technological hurdles are ascertained during research and developments. It is necessary to present a general overview of recent developments and challenges in this field. In this review paper, three aspects of advances in IL pretreatment are critically analyzed: biocompatible ILs, protic acidic ILs and combinatory pretreatments. [ABSTRACT FROM AUTHOR]